The Pamirs, among the world’s highest mountains in Central Asia, are one of homelands with the most extreme high altitudefor several ethnic groups. The settlement history of modern humans on the Pamirs remains still opaque. Herein, we havesequenced the mitochondrial DNA (mtDNA) genomes of 382 individuals belonging to eight populations from the Pamirsand the surrounding lowlands in Central Asia. We construct the Central Asian (including both highlanders and lowlanders)mtDNA haplogroup tree at the highest resolution. All the matrilineal components are assigned into the defined mtDNAhaplogroups in East and West Eurasians. No basal lineages that directly emanate from the Eurasian foundermacrohaplogroups M, N, and R are found. Our data support the origin of Central Asian being the result of East–WestEurasian admixture. The coalescence ages for more than 93% mtDNA lineages in Central Asians are dated after the lastglacial maximum (LGM). The post-LGM and/or later dispersals/admixtures play dominant roles in shaping the maternalgene pool of Central Asians. More importantly, our analyses reveal the mtDNA heterogeneity in the Pamir highlanders, notonly between the Turkic Kyrgyz and the Indo-European Tajik groups, but also among three highland Tajiks. No evidencesupports positive selection or relaxation of selective constraints in the mtDNAs of highlanders as compared to that oflowlanders. Our results suggest a complex history for the peopling of Pamirs by multiple waves of migrations from variousgenetic resources during different time scales.